Assessment of Histopathological Alterations and Oxidative Stress in the Liver and Kidney of Male Rats following Exposure to Aluminum Chloride

The study aims to investigate the residual and histopathological effects of chronic aluminum chloride (AlCl3) toxicity in the kidney and liver of male rats. After 30-, 60-, and 90-day exposure period, analyses were conducted to assess the toxicity in the kidney and liver. The results showed that the concentration of AlCl3 in the kidney and liver increased significantly in 30-, 60-, and 90-day periods. The effects of oxidative stress on the kidneys and liver were dose- and time-dependent. Levels of malondialdehyde (MDA) significantly increased when exposed to AlCl3 groups. Conversely, the activity of antioxidant parameters, including reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD), significantly decreased in the AlCl3 exposed groups, indicating compromised oxidant mechanisms. Both the kidney and liver exhibited severe tissue damage, including necrosis, fibrosis, and inflammatory cell infiltration, in rats exposed to AlCl3. Kidney sections showed hyperplasia of the epithelial cells lining the renal tubules, resembling finger-like structures. Liver sections displayed severe lobular hyperplasia and an increase in mitotic figures. Our study suggests that AlCl3 has a detrimental impact on these vital organs and emphasizes the importance of monitoring and mitigating aluminum exposure, particularly where it is present in high concentration.


Introduction
Aluminum (Al) is one of the most abundant elements on Earth and is commonly found in various natural and industrial sources.While aluminum is relatively inert in its elemental form, it can become toxic when present in soluble or bioavailable forms.Human exposure to aluminum compounds has increased over the years, primarily due to its presence in a wide range of products, such as food additives, drinking water, antacids, and cookware.It is the third most abundant metal in the Earth's crust, present in large quantities in soil and water [1].
Aluminum compounds are ingested through the consumption of food and beverages; they are commonly used as food additives to improve the texture and appearance of processed foods.Tese compounds can be found in baking powder, self-rising four, cake mixes, and some processed cheeses.Aluminum can enter the food supply through water sources, as it is sometimes used in water treatment processes [2,3].Tis can lead to aluminum contamination in drinking water and, subsequently, in food and beverages prepared with that water.In some regions, naturally occurring aluminum in soil and rock can leach into groundwater and surface water, leading to elevated aluminum levels in drinking water [4].Aluminum-based coagulants, such as aluminum sulfate, are used in water treatment to clarify water by removing impurities.While this is an essential process for water purifcation, residual aluminum levels in treated water can vary and potentially pose health concerns when consumed over a long period.
Moreover, some vaccines contain aluminum compounds as adjuvants to enhance the body's immune response to the vaccine.Tese include certain vaccines used in childhood immunization programs.Aluminum hydroxide is a common ingredient in antacids and other over-the-counter medications used to treat heartburn, indigestion, and acid refux.Prolonged use of such medications can lead to aluminum intake [5].Among children, authors found that vaccine-associated aluminum exposure is in favor of those children would be more than two and half times more likely to develop persistent asthma than those without this exposure [6].
It is important to note that the overall impact of aluminum exposure from these sources can vary signifcantly.While acute exposure to aluminum is generally not a cause for concern, chronic exposure, especially at high levels, may be associated with health risks, including hepatotoxicity, neurotoxicity, and potential links to conditions such as Alzheimer's disease [3,7,8].
Regulatory agencies, such as the World Health Organization and the U.S. Environmental Protection Agency, provide guidelines and regulations to address aluminum levels in drinking water and food additives, aiming to minimize potential health risks associated with aluminum exposure.Te typical concentration of airborne aluminum ranges from 0.0005 μg/m 3 in Antarctica to over 1 μg/m³ in industrialized regions [4].In natural waters, dissolved aluminum levels (at pH 7) typically range from 0.001 to 0.05 mg/L but can elevate to 0.5-1 mg/L in acidic or organically rich waters.With an average adult dietary intake of 5 mg/day of aluminum from food and a drinking water aluminum concentration of 0.1 mg/L, drinking water accounts for approximately 4% of total oral aluminum exposure.Generally, the contribution of air to overall aluminum exposure is minimal [4].
Globally, data on aluminum intake vary signifcantly due to multiple factors.For instance, adult dietary aluminum intake ranges from 7.1-8.2mg/day in the USA to 4.5 mg/day in Japan.Children's intake is reported at 0.8 mg/day in Germany, while in the United Kingdom and in the USA, it varies from 0.03 to 0.7 mg/day.In Germany, aluminum levels in public water supplies average 0.01 mg/l in the western region, but 2.7% of supplies in the eastern region exceed 0.2 mg/l [4].Cosmetics are deemed safe with aluminum concentrations of 2.65% in toothpaste and 0.77% in lipstick [9], and vaccines licensed in the USA are considered safe when containing aluminum ranging from 0.85-0.125mg per dose [10].
In the small intestine, aluminum chloride is rapidly converted to insoluble poorly absorbed basic aluminum salts, consisting of a mixture of hydrated aluminum oxide, oxyaluminum hydroxide, various basic aluminum carbonates, and aluminum soaps [11].Accumulation of aluminum in the brain has been associated with neurotoxicity and is of particular concern regarding its potential role in neurodegenerative diseases such as Alzheimer's disease.While the exact mechanisms are not fully understood, aluminum can induce oxidative stress and may contribute to the development or progression of neurological disorders [7,12].Chronic exposure to aluminum can have adverse efects on bone health as aluminum interferes with bone mineralization and can lead to bone demineralization, potentially contributing to conditions such as osteoporosis and osteomalacia [13,14].Te kidneys play a critical role in fltering and excreting aluminum from the body.Chronic exposure to aluminum can lead to renal dysfunction and damage, impairing the kidney's ability to flter waste products efectively [15,16].Inhalation of aluminum dust or ingestion of large amounts of aluminum compounds can lead to respiratory and gastrointestinal issues.However, these efects typically result from acute exposure to high levels, such as in occupational settings [4,17].
It is important to note that the potential health efects of aluminum exposure are a subject of ongoing research and debate.While there is evidence of aluminum's toxic potential, it is essential to consider the specifc source, duration, and concentration of exposure, as well as individual susceptibility.
Te objective of this study is to determine the residual, histopathological, and oxidative efects of chronic aluminum chloride toxicity on the kidney and liver in rats.

Experimental Animals.
A total of 90 male Wistar rats, aged between 4 and 9 weeks and weighing between 200 and 300 g, were obtained from the National Center for Drug Control and Research in Baghdad (Iraq).Tey were housed at animal house of the Veterinary Medicine College in Baghdad (Iraq) for 2 weeks before starting the experiment by rearing in separated, cleaned, and disinfected cages, and they were fed on commercial assorted pellets and clean water for drinking.All researchers are obligated to ensure the wellbeing of animals in their care, in strict adherence to the highest standards, and in accordance with Baghdad University laws, regulatory guidelines, and humane principles.In our study, the local animal welfare committee approved the current protocol.
Subsequently, the rats were randomly divided into three groups, with 30 per group.Te frst group of rats (GI: control) served as the controls and received ad libitum distilled water and standard diet.Te second group (GII: 100 mg/kg) had received orally the dose of 100 mg/kg bw. of AlCl 3 for 30, 60, and 90 days.Also, the third group (GIII: 200 mg/kg) had received orally the dose of 200 mg/kg bw. of AlCl 3 for 30, 60, and 90 days.Body weight and food intake were recorded every 6-7 days (Supplementary Table 1), and at the end of the experimental period (30, 60, or 90 days), the 2 Journal of Toxicology animals in diferent groups were sacrifced by cervical decapitation to avoid stress conditions.Te liver and the kidney were quickly excised, rinsed in ice-cold physiological saline, weighed, and then divided into parts: one for homogenization in the appropriate bufer at 10% (w/v) as indicated in the procedure's measurement of each parameter.Te supernatant aliquots were stored at −80 °C and used for biochemical assays, one was kept in formalin for histopathological analysis, and the last part was kept as such and put together at −80 °C until needed.

Determination of Aluminum in the Tissue Samples by
Flameless Atomic Absorption Spectrophotometer.Te initial standard solution for the desired element was 1000 μg/ml in 2% HNO 3 .Te trace element (aluminum) was determined using the fameless atomic absorption spectrophotometer method using the graphite furnace (GFAAS) technology.Four standard solutions (1000 μg/ml, 100 μg/ml, 10 μg/ml, and 1 μg/ml) were obtained by diluting the initial standard stock solution.A sequence of concentrations starting at the highest level and descending to the amounts necessary for the calibration curve's performance must be prepared.Te sample preparation is conducted by digestion of the animal tissue method [19].One gram of liver and kidney tissues was weighed and put into a 100 ml conical fask.Ten, fve milliliters of concentrated nitric acid (HNO 3 70%) and one milliliter of perchloric acid (HClO 4 ) were added, and the sample was allowed to sit at room temperature for one hour.Samples were placed on a heated plate at 100 °C until violet vapors started to form, at which point, the temperature was increased to 150-200 °C until white fumes started to appear.Te samples were fltered using paper (0.45), and the volume was adjusted with distilled water that had been acidifed with 1% HNO 3 up to the point when the remaining solution, which was pale yellow, indicated that the digestion process had been completed (25 ml).Results were read using the graphite furnace technique of atomic absorption spectrophotometer.

Determination of Oxidant and Antioxidant Parameters by ELISA (Enzyme-Linked Immunosorbent Assay).
Te double antibody sandwich technique of the ELISA was performed according to the My BioSource manufacturer's recommendation kits.Te preparation of tissue homogenates varies depending upon the tissue type.Tissues were collected and weighed before homogenization, minced to small pieces, and homogenized with a PBS (usually 10 mg tissue to 100 μl PBS.).Te homogenate is then centrifuged at 1000 × g (3000 rpm) for 20 minutes.Te supernatant was collected carefully, and immediately samples were stored at −80 °C until use.
Te ELISA analytical biochemical technique of the (MBS738685) kit for MDA, the (MBS701908) kit for catalase (CAT), the (MBS036924) kit for superoxide dismutase (SOD), and the (MBS2700076) kit for glutathione (GSH) is based on the enzyme-linked immunosorbent assay, which is to measure antigens by fxing antibodies associated with enzymes on them.Te principle of this technique is based on the visualization of an antigen-antibody reaction using a colorimetric enzymatic reaction.Te enzyme, previously coupled to the antibody, catalyzes a colored chemical reaction which transforms its substrate into a compound.After removing any unbound substances, a biotinconjugated specifc antibody is added to the wells.After washing, avidin-conjugated horseradish peroxidase (HRP) is added to the wells.Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and the color develops in proportion to the amount of molecules bound in the initial step.Te color development is stopped, and the intensity of the color is measured by spectrophotometer (Cecil, France) at 450 nm in a microplate reader (Biotech, USA).

Histopathological Study.
Liver and kidney samples, intended for histological examination, were processed overnight for dehydration, clearing, and impregnation using an automatic tissue processor (Sakura, Japan).Te specimens were embedded in parafn blocks using an embedding station (Sakura, Japan) and serial sections of 5 µm thickness were cut using a microtome (ModelRM2245, Leica Biosystems, Wetzlar, Germany) and stained by hematoxylin/ eosin as described by Bancroft and Gamble [16].Once dried, the sections were observed under a light microscope (Imager A2, Zeiss, Gottingen, Germany) at a magnifcation of 400× and photographed using a digital camera.

Statistical Analysis. Te Statistical Analysis System
program was used to detect the efect of diferent factors in study parameters (comparing the experimental group with the control group, each one containing 30 animals) [20].Least signifcant diference (LSD) test using analysis of variance (ANOVA).Results were presented as mean ± the standard deviation.Te level of signifcance used in the analysis was set at p ≤ 0.05, indicating a statistically significant diference between groups.

Residual of AlCl 3 in the Liver and Kidney
. Te determination by fameless atomic absorption of aluminum in the kidney and liver for toxicological monitoring of rats treated with the two diferent concentrations (100 and 200 mg/kg) is illustrated in Tables 1 and 2, respectively."An accumulation of aluminum was observed in the kidneys of treated rats, which signifcantly increased (p ≤ 0.05) with the duration of exposure at a dose of 100 mg/kg.Te aluminum concentration ranged from 1.88 ± 0.30 ppm during the frst 30 days to 8.08 ± 0.07 ppm after 90 days of exposure.In addition, the aluminum levels increased with higher concentrations of aluminum exposure across all study periods (30, 60, and 90 days) compared to the control group (0.034 ± 0.01 ppm, 0.037 ± 0.01 ppm, and 0.033 ± 0.01 ppm, respectively) (Table 1)."Te exposure at 200 mg/kg is more harmful and signifcant increase (p ≤ 0.05), ranging from 2.91 ± 0.01 ppm during the frst 30 days to 17.82 ± 0.11 ppm after 90 days of exposure and with increasing concentration of aluminum exposure during all periods studied 30, 60, and 90 days compared to control (Table 1).
Tis observation is consistent with the previous research study indicating that aluminum, when ingested, can accumulate in various tissues, especially the kidney, which plays a crucial role in fltering and excreting aluminum from the body.Such studies indicate that the oral bioavailability of aluminum from dietary sources is projected to be somewhat lower or similar compared to previous research studies focused on aluminum exposure through water, in both human and rat studies [2].Te prolonged exposure in this study likely allowed for the gradual accumulation of aluminum in these organs.
Aluminum treatment led to a signifcant decrease (p ≤ 0.01) in GSH in both the kidney (GII: 7.75 ± 0.03, 6.83 ± 0.05, and 6.19 ± 0.03 and GIII: 4.42 ± 0.41, 3.53 ± 0.44, and 2.78 ± 0.44) (Figure 1) and the liver (GII: 41. 16 2) compared to the control group in the kidney (GI: 8.06 ± 0.70, 9.03 ± 0.42, and   2).GSH is secreted from hepatocytes into the bile canalicular spaces.In the kidneys, GSH is degraded by membranous c-GT and other peptidases.Tis relationship explains the positive correlation of GSH levels found in the kidneys and liver of rats exposed to aluminum.Tese fndings suggest that chronic AlCl 3 exposure induces oxidative stress in the kidney and liver, compromising antioxidant defense mechanisms and leading to cellular damage, as confrmed by our fndings.Our results are consistent with those reported by Bondy [12] and Savory [16].Tey found that even in the group receiving the lowest aluminum content, the efect of chronic AlCl 3 exposure on lipid peroxidation levels was pronounced, with a highly signifcant diference.

Histopathological Efects of Aluminum Chloride in the
Kidney and Liver.Chronic exposure to aluminum chloride can lead to histopathological changes in the kidney (Figure 3) and liver (Figure 4) architectures, which are observable.Tese changes include hepatic infammation, potential hepatic fbrosis, altered hepatocyte architecture, the formation of granulomas or fatty changes, tubular damage in the kidney, interstitial infammation, glomerular alterations, and possibly fbrosis in renal tissues.
In our study, both the kidney and liver exhibited severe tissue damage in rats exposed to both 100 mg/kg and mg/kg of AlCl 3 over 30 (A), 60 (B), and 90 days (C).Kidney sections showed hyperplasia of the epithelial cells lining the renal tubules, which resembled fnger-like structures.Liver sections displayed severe lobular hyperplasia and an increase in mitotic fgures.Our results are consistent with previous studies [3,14].
Hyperplasia of the epithelial cells lining the renal tubules refers to an increase in the number of cells in the kidney, leading to enlargement of the afected area.Tis description likely refers to the architectural appearance of hyperplastic tubular cells.Normally, these cells are somewhat uniform, but with hyperplasia, they can proliferate excessively and irregularly, potentially forming projections or expansions that resemble fngers.
Liver hyperplasia refers to an increase in the number of liver cells, leading to the enlargement of the organ.It is characterized by the difuse transformation of the liver architecture into small nodules.Tis condition occurred due to chronic exposure to AlCl 3 , which afects the liver's ability to regenerate and respond to injury or increased functional demands.We found that animals in the control groups (GI) exhibited normal histological structure (Figures 3 and 4).In contrast, animals in GII, treated with 100 mg/kg bw AlCl 3 for 30, 60, and especially 90 days, showed marked atrophy of the glomerular tuft with thickening of Bowman's capsule and cystic dilation in the renal tubules (Figure 3).In the liver, there was marked thickening in the central vein, likely due to fbrosis and infltration of infammatory cells (Figure 4).
Animals in GIII, treated with 200 mg/kg bw AlCl3 for 30, 60, and especially 90 days, exhibited severe collapse of the glomerular tuft with thickening of Bowman's capsule and hyperplasia of the epithelial cells lining the renal tubules, forming fnger-like structures (Figure 3).Liver sections showed severe lobular hyperplasia (Figure 4).

Discussion
Te fndings concerning the accumulation of AlCl 3 in the kidney and liver and its potential role in aluminum poisoning have garnered signifcant interest in recent years.Aluminum is known to persist in the environment and can readily accumulate in various body tissues, posing potential harm to target organs such as the liver, kidneys, spleen, heart, and brain.Tis phenomenon has been supported by several studies.Many studies have highlighted the persistence of AlCl 3 in the body and its tendency to accumulate in critical organs [21].Other researchers have emphasized aluminum's potential to accumulate in various tissues, contributing to adverse efects on the body [22,23].Yu et al. observed that within the initial eight weeks of aluminum exposure, the liver and kidneys primarily accumulate aluminum [24].Tis fnding underscores how quickly aluminum can be sequestered by these organs.
Te kidney tends to accumulate higher levels of aluminum than the liver, depending on the dose and exposure duration, which aligns with the fact that the kidney plays a pivotal role in eliminating aluminum from the body.Approximately 95% of aluminum elimination occurs through urine, while the biliary pathway contributes only about 2% to total aluminum excretion [25].Tis fnding is consistent with the concept that kidney disease can impede aluminum removal, potentially leading to more pronounced aluminum accumulation in the kidneys than in the liver [26][27][28].
Tese fndings collectively underscore the signifcance of understanding the dynamics of aluminum accumulation in vital organs, particularly the kidney and liver, and its potential health implications.
Te vulnerability of vital organs, such as the liver and kidneys, to metal poisoning is well documented.Aluminum has shown to induce oxidative stress in these tissues, leading to adverse efects on cellular and tissue health.Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them with antioxidants.In the case of aluminum exposure, it has been observed that aluminum  can reduce the activity of the glutathione-synthase enzyme, resulting in decreased levels of glutathione (GSH), an essential antioxidant in the body.Consequently, the antioxidant defense mechanisms in tissues become compromised.
Other studies provide valuable insights into the mechanisms by which aluminum induces oxidative stress in tissues [29,30].Aluminum may inhibit NADPH-generating enzymes such as glucose 6-phosphate dehydrogenase, which are crucial for GSH regeneration [31].Moreover, our fndings showed an increase in malondialdehyde (MDA) levels and a decrease in the activities and levels of antioxidant molecules, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), both in the kidney and liver.Tese fndings suggest a clear link between aluminum exposure, oxidative stress, and the body's antioxidant defense mechanisms.Te rise in MDA levels in the kidney and liver indicates lipid peroxidation, which is a hallmark of oxidative stress and confrms that these tissues are experiencing oxidative damage due to chronic aluminum exposure [32].
Te reduction in the levels and activities of antioxidant molecules such as SOD, CAT, and GSH is concerning.Tese molecules play a vital role in neutralizing reactive oxygen species (ROS) and protecting cells and tissues from oxidative damage.A decrease in their levels suggests that the body's natural defense mechanisms against oxidative stress are compromised.
Te observed efects are dose-and time-dependent, indicating that the extent of oxidative stress and impairment of antioxidant defenses increase with higher doses and longer exposure to aluminum.Tis fnding underscores the importance of considering both the concentration of aluminum and the duration of exposure when assessing its potential health efects.
Studies have demonstrated that aluminum (Al) salts accelerate the peroxidation of membrane lipids induced by Fe (II) salts, suggesting that Al+3 interacts directly with cell membranes [33].Al+3 ions subtly alter the membrane structure, enhancing iron's oxidative activity.Aluminum has been found to increase the rate of lipid peroxidation in various tissues such as the liver, kidney, testis, and brain of diferent animals [34][35][36].
Another study found that mice receiving aluminum treatment for three months showed higher levels of MDA, while GSH levels decreased [37].Yi-Fei et al. reported that high doses of Al(NO 3 ) 3 induced an increase in lipid peroxidation rates.Even after brief exposure to Al(NO 3 ) 3 , mice exhibited signs of oxidative stress, including elevated lipid peroxidation (LPO), decreased GSH levels, and inhibited SOD and CAT activity in the liver and kidney [38].In addition, the defciency of antioxidant enzymes contributes to increased lipid peroxidation, which in turn damages cells.
Histological examination of the liver and kidney in the context of aluminum toxicity often reveals specifc structural changes indicative of damage and dysfunction.Te fndings of our study, which include histological lesions in the liver and kidney, are consistent with recent research study.Tese results underscore the potential harm that chronic exposure to AlCl 3 can cause to vital organs [39].
Te histological lesions observed in the liver, such as difuse hepatocellular regeneration, central vein congestion, and sinusoidal congestion in the centrilobular zone, indicate liver damage.Aluminum exposure leading to centrilobular necrosis, characterized by the death of hepatocytes, particularly in the centrilobular region, is a mark of liver damage due to toxic insult [40].Chronic aluminum exposure can also result in fatty infltration of the liver, marked by the accumulation of fat droplets within hepatocytes, impairing liver function [41].In addition, chronic aluminum exposure may lead to possible liver fbrosis, the accumulation of excess connective tissues in the liver, which disrupts its architecture and impairs function [42].Such changes can disrupt normal liver function and may be associated with the accumulation of aluminum in the liver.
Te liver sustains damage in the form of difuse hepatocellular regeneration, central vein congestion, and sinusoidal congestion in the centrilobular zone.Tese structural changes indicate liver dysfunction, disrupted blood fow, and infammation in response to aluminum exposure.
In the kidney, observed congestion in the capillary tuft of the glomeruli, difuse vacuolar degeneration in epithelial cells, and focal cystic changes suggest kidney damage.Aluminum exposure can lead to glomerular changes, such as glomerulosclerosis, characterized by thickening and scarring of glomerular basement membranes [43].Chronic aluminum exposure may also result in interstitial fbrosis in the kidney, the accumulation of fbrous tissues in the spaces between tubules and blood vessels [44].Aluminum can cause injury to tubular epithelial cells in the kidney, marked by cellular swelling, degeneration, and loss of brush border [45].Tese histological alterations are consistent with the potential impact of chronic aluminum exposure on renal tissues.
Te histological changes observed in our study, supported by several other studies, refect the diverse efects of aluminum exposure on the liver and kidney.Tese fndings provide scientifc evidence for our observations and underscore the importance of further research to better understand the mechanisms and potential health consequences of aluminum toxicity in tissues.

Conclusions
Chronic aluminum toxicity exerts signifcant and detrimental efects on both the liver and kidney, involving oxidative stress and structural damage to these vital organs.Te combination of these factors highlights the potential health risks associated with prolonged exposure to aluminum compounds.
Tese fndings underscore the multifaceted and interconnected consequences of aluminum toxicity.Oxidative stress and structural damage in the liver and kidney are interlinked processes, where oxidative stress can exacerbate tissue damage and compromise organ function.Te adverse efects observed in both organs emphasize the critical need for monitoring and regulating aluminum exposure, especially in environments where it is prevalent, such as certain occupational settings and sources of environmental contamination.

Figure 1 :
Figure 1: Oxidant parameters in the kidney of rats treated by AlCl 3 .Results are expressed as mean ± standard deviation.Means with the same letter are not signifcantly diferent.Means with diferent big letters in the same column and small letters in the same row are signifcantly diferent ( * p ≤ 0.05).

Figure 2 :Figure 3 :Figure 4 :
Figure 2: Oxidant parameters in the liver of rats treated by AlCl 3 .Results are expressed as mean ± standard deviation.Means with the same letter are not signifcantly diferent.Means with diferent big letters in the same column and small letters in the same row are signifcantly diferent ( * P ≤ 0.05).

Table 1 :
Residue of aluminum chloride in the kidney for chronic toxicity in experimental groups.Results are expressed as mean (ppm) ± standard deviation.Means with the same letter are not signifcantly diferent.Means with diferent big letters in the same column and small letters in the same row are signifcantly diferent ( * p ≤ 0.05).

Table 2 :
Residue of aluminum chloride in the liver for chronic toxicity in experimental groups.
4esults are expressed as mean (ppm) ± standard deviation.Means with the same letter are not signifcantly diferent.Means with diferent big letters in the same column and small letters in the same row are signifcantly diferent ( * p ≤ 0.05).4Journal of Toxicology 10.02 ± 0.93) and the liver (GI: 44.33 ± 2.08, 44.36 ± 1.33, and 45.41 ± 1.21), with decreases observed over the treatment period (Figure